Probing the Role of Surface Residues in the Complex Formed between Cytochrome C Peroxidase and Cytochrome C

The “lock and key” ionic-interaction model1 for the protein electron transfer complex between cytochrome c (cytc) and cytochrome c peroxidase (ccp) has been tested. Complex formation between these two proteins was probed by constructing site-directed mutations on the surface of ccp and preparing a fluorescent derivative of ccp, magnesium-porphyrin ccp (Mgccp). Data from fluorescence titrations, steady-state kinetics, chromatography and competition binding experiments suggest that this model only partially accounts for the electrostatic elements of binding. Aspartic acid 37 on the ccp surface, as predicted, plays a significant role in complexation and activity; when changed to lysine a significant decrease in the activity and binding to cytc is observed. Similar surface mutations at positions 79 and 217 show less dramatic effects.